Accumulation of DNA damage alters microRNA gene transcription in Arabidopsis thaliana

Background MicroRNAs (miRNAs) and other epigenetic modifications play fundamental roles in all eukaryotic biological processes. DNA damage repair is a key process for maintaining the genomic integrity of different organisms exposed to diverse stresses. However, the reaction of miRNAs in the DNA damage repair process is unclear. Results In this study, we found that the simultaneous mutation of zinc finger DNA 3′-phosphoesterase (ZDP) and AP endonuclease 2 (APE2), two genes that play overlapping roles in active DNA demethylation and base excision repair (BER), led to genome-wide alteration of miRNAs. The transcripts of newly transcribed miRNA-encoding genes (MIRs) decreased significantly in zdp/ape2, indicating that the mutation of ZDP and APE2 affected the accumulation of miRNAs at the transcriptional level. In addition, the introduction of base damage with the DNA-alkylating reagent methyl methanesulfonate (MMS) accelerated the reduction of miRNAs in zdp/ape2. Further mutation of FORMAMIDOPYRIMIDINE DNA GLYCOSYLASE (FPG), a bifunctional DNA glycosylase/lyase, rescued the accumulation of miRNAs in zdp/ape2, suggesting that the accumulation of DNA damage repair intermediates induced the transcriptional repression of miRNAs. Conclusions Our investigation indicates that the accumulation of DNA damage repair intermediates inhibit miRNAs accumulation by inhibiting MIR transcriptions. Supplementary Information The online version contains supplementary material available at 10.1186/s12870-022-03951-9.


Background
The cellular DNA of living species is normally damaged by endogenous and exogenous genotoxins. DNA bases are particularly susceptible to oxidation mediated by reactive oxygen species (ROS) [1]. The most thoroughly examined oxidized base product is 7,8-dihydro-8-oxoguanine (8-oxoG), which is produced due to the lower redox potential of guanine [1,2]. Failure to remove 8-oxoG results in G-to-T mutations [1]. Due to their sessile and photoautotrophic properties, plants are vulnerable to be attacked by ROS derived from photosynthesis and defence responses to biotic and abiotic stress [3,4]. Base excision repair (BER) is essential for repairing a wide range of lesions, including alkylation, deamination, oxidation and apurinic/apyrimidinic (AP) site lesions resulting from spontaneous depurination or processing of blocked 3′-ends of single-strand breaks, and BER is involved in active DNA demethylation to maintain balanced DNA methylation patterns [5][6][7]. The BER pathway in Arabidopsis is initiated by DNA glycosylases, which remove modified bases by cleaving the N-glycosidic bond and generate an abasic site without disruption to the phosphate-sugar DNA backbone [8,9]. FORMAMIDOPYRIMIDINE DNA GLYCOSY-LASE (FPG) and 8-oxoguanine DNA GLYCOSYLASE 1 enzymes show glycosylase/lyase activities that initiate the repair of oxidized 8-oxoG in plants [10,11]. Subsequently, the abasic sites are processed by enzymes with AP lyase activity or by AP endonucleases, producing either unconventional 3′-phospho-α,β-unsaturated aldehyde (3′-PUA) or 3′-phosphate (3′-P) and 5′-hydroxyl (OH) termini, or by cleaving the 5′ DNA backbone to generate 3′-OH and 5′-deoxyribose-5-phosphate (5′-dRP) ends [12][13][14][15]. Unconventional 3′-PUA or 3′-P and 5′-dRP ends need to be converted to conventional 3′-OH and 5′-P termini to enable subsequent polymerization and ligation. However, the molecular mechanism of BER in plants is unclear.
Plant miRNAs are 21-24 nucleotide (nt) small RNAs (sRNAs) that control development, immunity, metabolism, and other biological processes [32]. The transcription of miRNA-encoding genes (MIRs) and processing of primary miRNAs (pri-miRNAs) are coupled in the nucleus [33][34][35]. Pri-miRNAs are processed into stemloop precursors and then into miRNA/miRNA* duplexes via the Dicing complex [36,37]. Mature miRNA duplexes are mainly loaded into ARGONAUTE 1 (AGO1), which induces the transcriptional or posttranscriptional repression of target genes [38]. miRNA responses to DNA damage and the regulatory roles played by miRNAs in DNA damage repair and the DDR have been frequently reported in mammals [39]. For instance, miR-421 and miR-100 has been reported to suppress ATM expression by targeting the 3′ UTR of ATM transcripts [40,41]. The ATM kinase induced miRNA biogenesis by increasing pri-miRNA processing in mouse embryonic fibroblasts [42]. Moreover, deletion of Dicer in the developing mouse cerebellum resulted in accumulation of DNA damage [43]. In addition, the transcription factors E2F and Myc induced the transcription of miR-17-92, which was then posttranscriptionally inhibited by miR-17-92 in return, forming a feedback loop in a cancer network [44]. However, the roles of miRNAs in plant DNA damage repair and the DDR are unknown. The sRNA and degradome sequencing data analysis revealed that XPB2, a DNA repair helicase, was targeted by tae-miR1122c-3p in male sterile wheat lines [45]. MRE11, the gene encoding a DNA repair and meiosis protein, was putatively targeted by miR5261 in Citrus sinensis [46]. Based on recent studies, an interrelation between redox balance, the DDR, and miRNAs has been proposed [47]. However, studies on miRNA responses to DNA damage are scarce and preliminarily data have been primarily obtained via sRNA sequencing analysis. Through deep-sequencing profiling, 58 miR-NAs responding to DNA damage and 41 corresponding potential target genes related to DNA repair have been predicted [48]. In summary, the miRNA response to DNA damage remains elusive in plants.
The simultaneous mutation of ZDP and APE2 results in severe developmental phenotypes, including retarded root growth and slightly serrated leaves [16], which implies that miRNAs may be differentially expressed in zdp/ape2. In this study, we studied the reaction of plant miRNAs in response to DNA damage. We identified a genome-wide alteration of the miRNA population in zdp-1/ape2-2 mutant plants. The decrease in miRNAs was caused by reduced MIR transcription in zdp/ape2 mutant plants. We then found that the accumulation of DNA damage repair intermediates induced the transcriptional repression of MIRs. Our observations thus reveal that plant miRNAs react to DNA damage.
The decreased accumulation of pri-miRNAs and miRNAs in zdp/ape2 double mutant plants may have been caused by inhibited transcription of MIRs. To assess this possibility, we crossed zdp-1/ape2-2 mutant with a GUS reporter line under the control of the MIR159b promoter (pMIR159b::GUS) and obtained homozygous pMIR159b::GUS in zdp-1/ape2-2 plants [57]. GUS staining revealed that GUS activity was lower in pMIR159b::GUS in zdp-1/ape2-2 than that in pMIR159b::GUS in Col-0 plants (Fig. 2b). In addition, RT-qPCR analysis showed that the relative expression of GUS transcripts decreased by ~ 53.1% in the pMIR159b::GUS in zdp-1/ape2-2 compared with pMIR159b::GUS in Col-0 plants (Fig. 2c, Supplementary Table S6), which indicates that dysfunctional ZDP and APE2 led to the decrease in the transcription of MIRs. To confirm the positive role of ZDP and APE2 on MIR transcription, newly transcribed RNA transcripts were detected by nuclear run-on assays. RT-qPCR assays revealed that the abundance of newly transcribed MIR163, MIR165a, and MIR171a transcripts decreased significantly in the zdp-1/ape2-2 mutant (Fig. 2d, Supplementary Table S6). These results suggest that dysfunction of ZDP and APE2 decrease the transcription of MIRs, which leads to the reduced accumulation of pri-miRNAs and mature miRNAs.

Dysregulated DNA damage repair reduces MIR transcription
We then continued to determine the underlying mechanism by which the dysfunction of ZDP and APE2 decreases MIR transcription. As ZDP and APE2 play dual roles in active DNA demethylation and DNA damage repair [16,26], the decreased transcription of MIRs in ZDP and APE2 mutant plants may be caused by the altered accumulation of DNA methylation or damaged DNA. To test these possibilities, we measured the methylation levels on MIRs by analysing bisulfite sequencing data obtained from Col-0 and zdp-1/ape2-2 plants [16]. No significant difference in methylation level was found for MIR159b, MIR163, MIR165a, or MIR171a (Supplementary Fig. S4). Thus, the active DNA demethylation activity of ZDP and APE2 was likely not the cause of the transcriptional regulation of these MIRs.
As ZDP and APE2 play overlapping roles in BER [26], we wondered whether ZDP and APE2 affect MIR transcription through the DNA damage repair pathway. Small chemical alterations in DNA bases and single-strand DNA breaks are targeted by BER. We therefore treated Col-0 with the DNA alkylating reagent MMS to increase damaged bases. After 10 ppm MMS treatment, a mild reduction in pri-miR159b, pri-miR163, pri-miR165a, and pri-miR171a accumulation was observed in the Col-0 plants (Fig. 3a, Supplementary Table S7), indicating that DNA damage decreases MIR transcription. The zdp-1/ape2-2 mutant was also treated with 10 ppm MMS. RT-qPCR assays showed that the accumulation of pri-miRNAs in the MMS treated zdp-1/ape2-2 was significantly lower than that in the control zdp-1/ape2-2 mutant plants (Fig. 3a, Supplementary Table S7). Moreover, the accumulation of pri-miRNAs in MMS-treated zdp-1/ape2-2 decreased dramatically compared with that in MMS-treated Col-0 (Fig. 3a, Supplementary Table  S7). Moreover, the levels of mature miRNAs decreased significantly in the zdp-1/ape2-2 mutant after 10 ppm MMS treatment (Fig. 3b, Supplementary Table S7). These results indicate that the reduction in miRNAs in the week-old seedlings of Col-0, zdp-1/ape2-2, and zdp-1/ape2-3 plants. EF1α was used as the internal control. Error bars represent standard deviation calculated on the basis of 3 independent replicates. Statistically significant differences between different genotypes are indicated by different lower case letters (P < 0.05, one-way ANOVA per gene, performed separately). B GUS staining of samples harbouring a pMIR159b::GUS in Col-0 or zdp-1/ape2-2 background. Scale bar, 0.2 cm. C GUS transcript level was determined by RT-qPCR in samples harbouring a pMIR159b::GUS in Col-0 and zdp-1/ape2-2 background. EF1α was used as the internal control. Asterisks indicate significant differences in GUS expressions between plants with Col-0 and zdp-1/ape2-2 mutant background (Student's t test, **P < 0.01). D Transcription rates of MIR163, MIR165a, and MIR171a in Col-0 and zdp-1/ape2-2 plants, as measured by nuclear run-on assay and RT-qPCR. EF1α was used as the internal control. Asterisks indicate significant differences in relative expression level of MIR transcripts between Col-0 and zdp-1/ape2-2 plants (Student's t test, **P < 0.01) zdp/ape2 mutant can be attributed to the malfunction of ZDP and APE2 in DNA damage repair pathway.

Fig. 3 MIR transcription is inhibited by MMS treatment in zdp/ape2 mutants. A RT-qPCR detection of the relative expression levels of pri-miRNAs in
Col-0 and zdp-1/ape2-2 mutant plants treated with 0 ppm or 10 ppm MMS. EF1α was used as the internal control. Error bars represent the standard deviation calculated on the basis of 3 independent replicates. Statistically significant differences between different genotypes are indicated by different lower-case letters (P < 0.05, one-way ANOVA per gene, performed separately). B RT-qPCR detection of the accumulation of miRNAs in Col-0 and zdp-1/ape2-2 plants treated with 0 ppm or 10 ppm MMS. U6 was used as the internal control. Error bars represent the standard deviation calculated from 3 independent replicates. Statistically significant differences between different genotypes are indicated by different lower-case letters (P < 0.05, one-way ANOVA per gene, performed separately) treated with 0 or 10 ppm MMS. The relative expression level of the pri-miRNAs in the fpg-1 mutant was similar to that in the Col-0 (Fig. 4, Supplementary Table S8). In addition, the accumulation of pri-miRNAs were comparable between fpg-1/zdp-1/ape2-2, fpg-1/zdp-1/ape2-3 and fpg-1 plants with or without MMS treatment (Fig. 4,  Supplementary Table S8). A previous study suggested that the introduction of mutation in FPG strongly recovered the developmental defects in zdp/ape2 mutant [26]. Taken together, these results indicate that FPG dysregulation prevents the accumulation of DNA repair intermediates in zdp/ape2 mutant, and that the accumulation of 3′-blocked DNA repair intermediates decrease the plant miRNA transcription.

Discussion
DNA damage repair plays crucial roles in all species to maintain genome integrity. However, the molecular mechanisms of miRNA responses to DNA damage repair and the DDR in plants are unclear. In this study, we determined the reaction of miRNAs to DNA damage and found that the decrease in MIR transcription was accompanied by the accumulation of DNA damage repair intermediates (Fig. 5). Fig. 4 The mutation of FPG on zdp/ape2 background rescues the accumulation of pri-miRNAs. RT-qPCR detection of the relative expression levels of pri-miRNAs in Col-0, zdp-1/ape2-2, fpg-1, fpg-1/zdp-1/ape2-2, and fpg-1/zdp-1/ape2-3 plants treated with 0 ppm or 10 ppm MMS. EF1α was used as the internal control. Error bars represent standard deviation calculated from 3 independent replicates. Statistically significant differences between different genotypes are indicated by different lower-case letters (P < 0.05, one-way ANOVA per gene, performed separately) miRNAs react to DNA damage stress in plants. DNA double-strand breaks modulate the expression of multiple miRNAs through several mechanisms in mammals [42,[58][59][60]. A total of 150 miRNAs show differential expression levels in bleomycin-treated rice roots [48]. Through small RNA sequencing, we observed a genome-wide alteration of miRNAs in zdp-1/ape2-2 mutant plants. Moreover, the total expression levels of downregulated miRNAs accounted for the majority of the total miRNAs that had accumulated. Abiotic and biotic stress can induce an oxidative burst that damages DNA [61,62]. Although 4 miRNAs (miR159b, miR163, miR165a, and miR171a) detected in this study, the levels of other miRNAs, namely, miR827 and miR399 family members (miR399a, miR399c, miR399d, and miR399f ), were profoundly reduced in the zdp-1/ape2-2 mutant. The common features of these miRNAs include involvement in development regulation and biotic and abiotic stress responses [51][52][53][54][63][64][65][66][67]. Besides, the developmental defects displayed by mutated DNA damage-related genes might be common in Arabidopsis [24,68]. In addition, we noticed that the abundance of miR843, miR845a, and miR866-5p, which are involved in mediating the genome dose balance by triggering the production of epigenetically activated sRNAs to target transposable elements [69,70], increased markedly in the zdp-1/ape2-2 mutant. Therefore, our study reveals a genome-wide alteration of miRNAs in response to DNA damage.
DNA damage affects MIR transcription in plants. Our results reveal that the simultaneous mutation of ZDP and APE2 affects miRNA biogenesis at the transcriptional level. Studies in mammals have revealed that miR192, miR194, and miR215 are transcriptionally activated by P53 [71,72]. Other transcription factors play major roles in the DDR, such as Myc and E2F, and induce the expression of several miRNAs in human cells [44]. The malfunction of ZDP and APE2 in BER leads to the accumulation of 3′-blocked DNA intermediates, activating the DDR [16,26]. The DDR is ultimately involved in the transcriptional regulation of multiple genes controlling cell cycle checkpoints, DNA repair and programmed cell death [24]. DNA-dependent RNA polymerase II-mediated MIR transcription is regulated in a sophisticated manner in plants [33,34]. Therefore, the transcription of MIR might be regulated by DNA damage and the DDR.
The accumulation of DNA repair intermediates inhibits MIR transcription. Unrepaired DNA lesions and intermediates initiate the DDR [24][25][26]. Genes involved in the DDR, including RAD51, BRCA1, MRE11 and WEE11, were activated in zdp/ape2 mutant under normal conditions [16]. Arabidopsis APE2 carries an GRF-type zinc finger domain [16], and shows high sequence similarity to human APE1 [73]. hAPE1 functions in damaged DNA/ The FPG-mediated BER pathway initiated under oxidative stress, generating blocked 3′-P end repair intermediates. In WT plants, ZDP and APE2 converted 3′-P end to 3′-OH end to allow subsequent progress in base repair. In zdp/ape2 mutant, 3′-P blocked repair intermediates accumulate and lead to the inhibition of MIR transcription, which causes reduced accumulation of pri-miRNAs and miRNAs. The starburst indicates an oxidized base RNA repair, and the downregulation of hAPE1 is accompanied by alterations in miRNA expression [74,75]. Thus, the decrease in miRNA expression in the zdp/ape2 mutant in our study might have been related to the direct function of ZDP and APE2 on MIR transcription regulation or the indirect regulation by the DDR invoked by the accumulation of 3′-blocked DNA repair intermediates. Treatment with MMS exacerbated the decrease in miR-NAs in zdp/ape2 mutant, and mutation in FPG, which initiates DNA damage repair, rescued miRNA expression in the zdp/ape2 mutant plants. These findings suggest that MIR transcription alterations react to the accumulation of DNA damage repair intermediates.
Animal miRNAs that respond to DNA damage can also modulate DNA damage in return [39,76]. The interrelationship between DNA damage and miRNA biogenesis is proposed, especially with respect to the feedback loop comprising miR-17-97 and the E2F and Myc transcription factors in a cancer network [44]. A few studies in plants have also indicated that changes in accumulation of miRNAs might regulate DNA damage in feedback loop mechanism [39,44,48]. The DNA repair helicase XPB2 has been predicted to be a target of miR166a-3p, and a putative suppressor of the stem-loop protein 1 Os04g42990 has been predicted to be a target of miR167d and miR167a-5p in rice [48]. Our observations show that the accumulation of miR166 and miR167 is reduced in zdp-1/ape2-2 mutant. These results suggest that plant miRNAs react to DNA damage and may form a feedback loop during the DDR.

Conclusions
Our investigation suggests that DNA damage repair intermediates regulate miRNA accumulation at transcriptional level.

RNA extraction
Seedlings harvested from MS medium were grounded into fine powder in liquid nitrogen, and mixed with Trizol reagent (Invitrogen, 15,596,018) for RNA isolation. The solution was mixed thoroughly and added 1/5 volume of chloroform for homogenization. After incubation at RT for 5 min, the sample was centrifuged at 12,000 rpm at 4 °C for 15 min. The supernatant was mixed with 2.2-fold volume of ethanol and stored at -20 °C overnight. The mixture was then centrifuged at 12,000 rpm at 4 °C for 15 min. The pellet was washed with 75% ethanol and dissolved with RNase-free H 2 O.

Small RNA library construction and analysis
Small RNA library was constructed as previously reported [78]. Briefly, total RNA was extracted from 2-week-old seedlings grown on 1/2 MS medium with Trizol reagent. 30 μg RNA was loaded on 15% urea-PAGE gel, and the small RNAs range from 18-30 nt were sliced from the gel. Small RNAs were recovered by soaking the smashed gel in 0.3 M NaCl overnight, followed by precipitation with ethanol. Small RNA libraries were constructed following instructions from NEBNex ® Small RNA library Prep Set for Illumina ® (NEB, E7300S). 4 small RNA libraries were constructed both for Col-0 and zdp-1/ape2-2 samples. The small RNA libraries were single-end sequenced on an Illumina HiSeq2500 platform.
Small RNAs sequences were processed with Cutadapt v3.4 [79] to remove sequencing adaptors and lowquality bases. Reads with length between 18 to 50 nt were retained for further analyses. Clean reads were mapped to the Arabidopsis genome (TAIR10 version) with SPORT v1.1.1 [80] and were annotated into noncoding RNA categories, including miRNA, tRNA, rRNA, siRNA, etc. To perform differential expression analysis of miRNA, clean reads were mapped to the Arabidopsis genome (TAIR10 version) with bowtie v1.3.0 [81] allowing no mismatches. Uniquely mapped reads were used to calculate miRNA counts with featureCounts v2.0.1 [82]. miRNA abundance was normolized to reads per million with the sum of 18-30 nt reads. Fold-change of RPM between Col-0 and zdp-1/ape2-2 was calculated for each miRNA. Fold-change ≥ 1.5 or ≤ -1.5 were used as threshold for differential expressed miRNAs. Correlation was calculated using Euclidean's distance matrix using PtR program in Trinity package [83].

RT-qPCR
Expression levels of pri-miRNAs, mRNAs, and miRNAs were examined by quantitative real-time PCR. Total RNA was extracted using Trizol reagent from 2-week-old seedlings grown on 1/2 MS medium. For mRNA reverse transcription, cDNA was synthesized in 20 μl reaction volumes using 1 μg DNase-I (NEB, M0303) treated total RNA and reversely transcribed with the Evo M-MLV Mix Kit (AIKERUI, AG11728). For miRNA reverse transcription, 1 ug of total RNA was digested with DNase I and poly (A) was added to the 3′ end by E. coli poly (A) Polymerase (NEB, M0276). The first-strand cDNAs were transcribed by M-MuLV reverse transcriptase (NEB, M0253). RT-qPCR was performed in 10 μl volumes containing 2 μl of 20-fold diluted cDNA, 5 μl of SYBR Green mix (Vazyme, Q311), and 0.2 μM of each primer. The analysis was performed in One-way PCR detection system (Invitrogen) using the following cycling conditions: initial denaturation at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. All data was normalized to EF1α. Primers used in RT-qPCR are listed in Supplementary Table S4. The relative fold change in the expression levels were calculated using the 2 −∆∆Ct method. All reactions were carried out in 3 biological replicates.

Northern blot
Northern blot was performed as described [35,84]. Total RNA was separated on 14% denaturing ureapolyacrylamine gels and run with 0.5 × TBE at 150 V. The gel was transferred to Hybond membrane NX (GE healthcare, RPN303T) at 14 V overnight. Chemical crosslink buffer was prepared as follows: 0.373 g N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (Sigma-Aldrich, E7550), 3 drops of 1 M HCl, 121 µl Methylimidazole (Sigma-Aldrich, M50834), and 12 mL RNase-free H 2 O. After chemical crosslink at 60 °C for 2 h and UV crosslink at 85 °C for 2 h, the membrane was preincubated with PerfectHyb ™ Plus Hybridization Buffer liquid (Sigma-Aldrich, H7033) for 30 min, then hybridized overnight at 37 °C with γ-32 P ATP (China isotope & radiation corporation) labelled DNA probes by T4-polynucleotide kinase (NEB, M0201S) for 4 h. After that, the membrane was washed with buffer contains 2 × SSC and 0.025% SDS. Auto-radiography of the membrane was performed using a Typhoon Scanner. Sequences of probes are listed in Supplementary Table S4.

GUS staining
GUS staining was performed with 2-week-old plants. Seedlings were immersed in GUS staining solution and incubated at 37 °C overnight. After staining, rinse the seedlings in 75% ethanol until the clear of chlorophyll. Pictures were taken under stereo microscope.